ADP/ATP Ratio Assay Kit (Bioluminescent) (ab65313)

The assay buffer has high concentration of detergent-hence Bradford assay is not recommended. We advise using the BCA assay for protein measurement when using this kit.
This assay is performed on cell lysates, so it would be difficult to label the cells with Hoechst after the assay.

Thank you for contacting us. Regarding your questions from yesterday:

1. Why is data C required after data B acquisition?

The two readings should be similar and perhaps data C is not necessary but the ATP signal may go down slightly while the ADP converting enzyme is prepared.

2. Why wait a few hours after adding the ATP Monitoring Enzyme and Nucleotide Releasing Buffer to the wells and before reading data A?

There may be bacterial contamination in the well or reagents that will produce some ATP signal, which should disappear after a few hours. Again, the wait may not be necessary if there is no contamination. (The note in step 3 about waiting a few hours should probably be at the start of step 3. There is no need to wait a few hours AFTER reading data A).

3. What is the best sample preparation protocol for pancreatic islet cells?

I recommend incubating each islet sample with 50ul of the Nucleotide Releasing Buffer, incubating for 5 minutes at room temperature with gentle shaking, and then adding this mixture to the ATP Monitoring Enzyme and Nucleotide Releasing Buffer mixture in the assay plate.

The signal should be fairly stable as long as the sample is protected from light. But we suggest measuring after 2 mins to make sure that no light is quenched. Waiting for 10 mins is not advisable since some light can be quenched during this time. For the specific samples in question, if there is no significant light by 2 mins, then most likely more sample should be used in the well.

We can recommend two protocols:
1. Prepare a single cell suspension from the tissues of interest with any method desired (cells have to stay intact). Then treat the cells as instructed in the protocols section for adherent cells: Remove culture medium/ buffer and treat cells with 50 μl of Nucleotide Releasing Buffer for 5 minutes at room temperature with gentle shaking. Transfer into luminometer plate. (page 8, 4b).
2. Chop the tissues to the finest possible size (keep cool). Then treat cells with 50 μl of Nucleotide Releasing Buffer for 5 minutes at room temperature with gentle shaking. Transfer into luminometer plate.
Please note that the tissue preparation needs to be optimised by the end user since an increased number of dead cells may affect the results.

Please see the following publication which may be of use to you:

Diguet N et al. Muscle creatine kinase deficiency triggers both actin depolymerization and desmin disorganization by advanced glycation end products in dilated cardiomyopathy. J Biol Chem 286:35007-19 (2011).

Protocol:
ATP and ADP/ATP ratios were measured using the bioluminescent ADP/ATP ratio assay kit from Abcam as indicated by the manufacturer with minor modifications. Briefly, cardiac tissue was frozen in liquid nitrogen and powdered with a mortar. Tissue powder was suspended in the provided lysis buffer (10 μl/mg of tissue powder) for 5 min exactly at room temperature, followed by centrifugation at 10,000 × g for 1 min to pellet insoluble material. We used 50 μl of supernatant per assay in a final volume of 100 μl. Luciferase units were converted to picomoles of the amount of ATP by plotting against a standard curve with serial dilution of ATP. Data were normalized by the amount of protein present in the supernatant as measured by the Bradford assay.

Thank you for your inquiry.

I am happy to confirm that ab65313 can be used with mouse liver tissue samples.

This kit can be used to measure the ADP/ATP Ratio, AMP is not measured.

If you are planning to use this kit with tissue samples, I would like to note that the preparation of the samples need to be optimized according to the sample type etc..

A single cell suspension has to be prepared from the tissue and this can then be treated as the adherent cells in the prototocol.

Another possibility is to cut the tissue in very small pieces and then incubate for 5 minutes at room temperature in Nucleotide releasing buffer.

I hope this information is helpful and wish you good luck for your results.

Yes, you are correct. Lower ATP readings would indicate a decrease in cell viability.

Ab65314 is recommended for cell viability. For media, use the same protocol as mentioned for the suspension cells in step 2.a.